The present invention relates to a new ATPase assay. This invention particularly relates to a new method for the detection and measurement of the amount of orthophosphate released by hydrolysis of ATP or any other phosphate containing molecule. More particularly, this invention relates to a new method for the measurement of the ATPase activity of the E1 helicase enzyme from the human papilloma virus (HPV).
HPV-associated disease
The human papillomaviruses (HPVS) are small DNA viruses that infect cells of the cutaneous and mucosal epidermis. Over 80 different HPV genotypes have been characterized. Some types, such as HPV-1, -2, -3, -4 and -10, cause cutaneous lesions known as warts or papillomas. These growths are benign and self-limiting, and are found on the hands and feet of 7-10% of the general population. Of greater medical concern are those HPV types that infect the anogenital tract. These genotypes are designated as either xe2x80x9clow-riskxe2x80x9d or xe2x80x9chigh-riskxe2x80x9d based on their correlation with malignant progression.
So-called low-risk HPVs are associated with genital warts, or condyloma acuminata. For instance, HPV types 6 and 11 are found in more than 90% of benign genital lesions, and very rarely associated with malignant transformation. However, they nonetheless represent a serious public health problem. Approximately 1% of sexually active adults in the U.S.A. have visible genital warts, but in many more cases the infection is sub-clinical. In fact, an estimated further 15% of people aged 15-49 display molecular evidence of HPV infection, in the form of viral DNA detectable by polymerase chain reaction (PCR) assay. Indeed, HPV is ranked as the most common sexually-transmitted viral agent in the U.S.A. and U.K., and its incidence is increasing steadily.
Infection with high-risk HPV types such as 16,18, 31 and 33, has been strongly linked to the development of anogenital malignancies, most notably cervical cancer. In fact, HPV types 16 and 18, while rarely found in benign genital lesions, are detectable in about 70% of all invasive carcinomas of the cervix. The link between HPV and anogenital cancer is well documentedxe2x80x94recent studies have found that almost 90% of cervical carcinomas contain HPV DNA.
In spite of the pervasiveness of HPV infection and its possibly life-threatening consequences, no virus-specific inhibitor has yet been described. Antiviral drug discovery for HPV has proven quite difficult thus far as a result of difficulties encountered in propagating the virus in the laboratory.
All current therapies for HPV infection rely on the non-specific destruction or removal of infected tissue. Accepted surgical procedures include the use of dry ice, liquid nitrogen, CO2 laser therapy, electrocautery or local excision. Various cytotoxic agents are also used to destroy tissue, such as salicylic acid, tricholoroacetic acid, podophyllin, colchicine, bleomycin and cantharidine.
While the risk of cancer makes these procedures the most prudent for the treatment of high-risk HPVs, less invasive treatments are being sought to manage the low-risk genotypes. Compounds that stimulate the immune system have been investigated with the goal of reproducing the spontaneous regression often seen with benign lesions. Imiquimod, such an immune response modifier, has recently passed clinical trials and been approved for treatment of HPV-associated genital warts.
Patients with genital warts often experience high recurrence ratesxe2x80x94usually 30-90%xe2x80x94following non-specific treatments such as surgery. Such poor efficiency is a result of the incomplete elimination of HPV DNA, or the presence of virus in normal-appearing tissue adjacent to the papilloma. Obviously, there is a substantial need for an effective, virus-specific therapy for HPV infection, which has thus far gone unmet.
Semi-conservative DNA replication is an intricate process mediated by many enzymes and accessory proteins. Helicases are enzymes that function during DNA replication, catalyzing the unwinding of duplex DNA ahead of the replication fork. They are very common in prokaryotic and eukaryotic cells, as well as most viruses. The exact mechanism by which helicases convert the binding and hydrolysis of ATP into mechanical energy to power the unwinding of DNA and their own simultaneous motion along the nucleic acid stand is still not completely understood.
The 72 kDa HPV E1 protein has been classified as a member of helicase superfamily III along with the T antigen of Simian Virus 40 (SV40 TAg), with which it is structurally and functionally homologous. E1 and Tag belong to a noteworthy subgroup of viral DNA helicases which have the ability to recognize and bind specific DNA sequences at the viral origin of replication (ori). Also, while most DNA helicases require a region of single-stranded DNA for entry, these proteins can initiate unwinding from completely double-stranded DNA, provided it contains an ori.
Human papillomaviruses contain approximately 8 kb of double-stranded circular DNA. In the basal cells of the epidermis, the genome is replicated and maintained extra-chromosomally at a steady-state level of about 20-100 copies per cell. High-level amplification of the genome only occurs once the cell has terminally differentiated and migrated to the upper layers of the epithelium.
In a cell-free DNA replication system, the E1 protein can direct origin-specific DNA replication by itself at sufficient concentrations, when provided with the full complement of host replication proteins including the DNA polymerase a primase enzyme. However, replication is greatly stimulated by the viral E2 protein, and at limiting concentrations of E1 the in vitro replication becomes completely E2-dependent. This is a consequence of E1 having a relatively low affinity for its DNA binding site. E2 helps to localize E1 to the origin by acting as an accessory protein . The E1 and E2 binding sites at the viral ori are in close proximity, falling within about 100 bp of each other. The carboxy terminus of E2 binds its palindromic site on DNA, while the amino terminus binds E1, thus bringing E1 to its binding site.
Recently, pharmaceutical companies have been able to substantially expand and accelerate their antiviral compound screening programs as a consequence of advances in molecular biology. Viruses are now routinely examined at the molecular level to find specific inhibitors of virus-encoded gene products.
For several viruses, enzymes such as polymerases, kinases and proteases have been targets for inhibition. In contrast, of the approximately 8 distinct proteins encoded by the HPV genome, the E1 helicase is the only one with enzymatic activity (Fields et a., 1996, Fields Virology, 3rd Ed. Lippincott-Raven, Philadelphia, Chap. 65 and refs. therein). E1 displays or-specific DNA-binding activity, E2-binding activity, ATPase activity, and DNA helicase activityxe2x80x94all of which can be assayed independently for potential inhibitors. In addition, it is the most highly conserved of all papillomavirus proteins, so an inhibitor of E1 would likely be effective against multiple HPV types.
High throughput screens are known that allow the discovery of inhibitors of the helicase activity of E1 (WO 99/157283, Nov. 11, 1999). Even though ATP is needed to drive E1 helicase activity and is included in the reaction, this helicase assay cannot be used to identify competitive inhibitors of E1 ATPase function. This is a direct result of very low Km of the ATPase, for example approximately 10 xcexcM for HPV-11 E1, and the fact that the helicase assay is routinely run with 300 xcexcm-1 mM ATP). A more sensitive assay must be developed if the ATP binding site of E1 is to be targeted for inhibition.
Helicase activity is virtually always associated with nucleoside triphosphatase activity (Matson et al., Ann. Rev. Biochem., 1990, 59, 289). Enzymatic ATP hydrolysis has been measured by a variety of methods, including colorimetric reactions; in all cases, enzymatic reactions are performed according to enzyme-specific protocols where reaction conditions are not dependent on the detection procedure (except for the inclusion of radiolabeled ATP). The detection procedure differs for the different assays in the following ways:
TLC:
The inclusion of [xcex1-33P] or [xcex3-33P] ATP in the substrate for an ATPase reaction results in the release of radiolabeled phosphate or ADP. Because of their different polarity, [33P]-labeled ATP, ADP and phosphate can also be separated by thin layer chromatography (Bronnikov et al., Anal. Biochem., 1983, 131, 69) in a running solvent (e.g. lithium chloride/formic acid). The two species migrate at different distances on a TLC plate based on their relative affinities for the polar mobile phase and non-polar solid phase. Results are analyzed by scintillation counting or Phosphorlmager analysis.
Although the TLC assay for quantification of released phosphate produces accurate data for ATPase activity and inhibition, it is unsuited for the mass-screening of potential inhibitors. The spotting and running of large numbers of TLC plates is time-consuming and labor-intensive. A method that lends itself to 96-well plate format and rapid quantification is needed if an ATPase assay is to be implemented in HTS format.
Charcoal:
ATP binds to charcoal but orthophosphate does not (Zimmerman et al. J. Biol. Chem. 1961, 236 (5), 1480). Thus if a reaction is run using xcex3-labeled ATP and charcoal is added, the starting material is adsorbed, but the product remains in solution. One can run this as a 96-well plate assay by filtering solutions through charcoal-containing filter plates, and counting the flow-through. This is not likely to be highly reproducible, and is not amenable to robotic screening.
Coupled-enzyme Assays:
There are a number of related procedures in which another reaction is carried out on the phosphate product by a second enzyme (Rieger et al., 1997, Anal. Biochem. 246, 86 and refs. contained therein). These assays are very useful for kinetic studies, because absorbance change is generated continuously over the course of the assay, so that the reaction course can be monitored without removing aliquots as necessary for the other methods above (the distinction between continuous and stop-time assays). These methods are not significantly more sensitive than the molybdate assay (below) however, and screening results would be further complicated by the possibility of false positives being inhibitors of the coupling enzyme.
Molybdate:
Ammonium molybdate forms a complex only with phosphate to form phosphomolybdate. Pyrophosphate, nucleotide triphosphates, or other phosphate-containing molecules resulting from the reaction do not interact with molybdenum oxides. Most of the colorimetric reactions are based the formation of a complex between phosphate and the molybdate ion in acid solution, followed by reduction or binding to dyes that form colored complexes. Many variations to these techniques have been introduced with the goal of increasing sensitivity and color stability, and decreasing the amount of spontaneous ATP hydrolysis that occurs during the color-developing incubation (Gonzalez-Romo et al., Anal. Biochem. 1992, 200, 235). For instance, the phosphomolybdate complex can be reduced by ascorbic acid to generate a blue molybdenum chromogen with maximum absorbance at 700 nm (Hergenrother et al., Anal. Biochem. 1997, 251, 45). Another method is based on the formation of a brilliant green complex with malachite green in an acid medium, which has a maximum absorbance at 650 nm (Moslen et al., Anal. Biochem. 1983, 131, 69).
In fact, the malachite green assay was previously evaluated as a potential test for the ATPase activity of E1, but was found to be unsuitable because it could not accurately detect concentrations of phosphate lower than 25 xcexcM. This presented a problem, because detection of competitive inhibitors is optimal at substrate concentrations below the Km of an enzyme. As previously mentioned, the Km (ATP) of the HPV-11 E1 ATPase has been shown to be about 10 xcexcM, so the E1 ATPase reaction is routinely carried out at around 1-10 xcexcM ATP. In addition, substrate consumption in an inhibition experiment is kept below 30%, so that substrate concentration remains essentially constant over the time of the reaction. The result is that 3 xcexcM is the maximum concentration of phosphate that is releasedxe2x80x94well below the 25 xcexcM detection limit of the malachite green assay.
All these colorimetric ATPase assays require a minimum ATP concentration of several hundred micromolar. The value of Km(ATP) for HPV-11 E1 being approximately 10 xcexcM (measured in the absence of DNA), thus to effectively screen for competitive inhibitors of E1 ATPase activity, one should perform assays using [ATP]xe2x89xa610 xcexcM.
Adsorption of Phosphomolybdate on Solid Support:
Phosphomolybdate is a large heteropolymolybdate, with a stoichiometry of [PMo12O40]3xe2x88x92. Because of its relatively low charge, it can be extracted from aqueous solution into organic solvents or adsorbed onto a hydrophobic surface such as Sephadex beads or nitrocellulose filters. Ohnishi et al. (J. Solid-Phase Biochem. 1976, 1(4), 287) and Ohnishi (Anal. Biochem. 1978, 86, 201) disclose a method for isolating the phosphomolybdate complex from solution by affinity chromatography on polyvinyl polypyrrolidone (PVPP) column. PVPP acts as a catalyst for the complexing reaction between PO4 and molybdenum and thereby selectively adsorbs the complex over other phosphate-containing molecules. Phosphate may be radioactively labeled and eluted from the column for counting of radioactivity. This method is limited by the fact that the labeled phosphate needs to be separated from the reaction mixture before counting. There remains a need for a robust method for phosphate determination that is amenable to a high throughput format.
Yoshimura et al. (Anal. Biochem. 1986, 58, 591) disclose a colorimetric micro-determination of molybdenum-blue by adsorbing the complex on Sephadex gel-phase. This procedure requires reduction of the complex prior to the adsorption and measures the phosphate concentration by direct absortiometry of the heteropoly acid concentrated in the gel phase. This procedure requires separation of the gel beads from the supernatant prior to measurement by colorimetry. Although this colorimetric method allows for detection of low concentrations of phosphate, it remains unsuitable for automation.
Scintillation Proximity Assay:
Hart et al. (Molec. Immunol. 1979, 16, 265) and Hart et al. (J. Nucl. Med. 1979, 20,1062) disclose a new method for immunoassay called xe2x80x9cscintillation proximity assayxe2x80x9d. This technology used scintillant latex particles coated with a ligand that specifically binds an organic reactant being investigated. All further applications of this technology with hydrophobic beads has relied on providing a specific ligand coated on the beads to bind specifically to a molecule.
U.S. Pat. No. 4,568,649 discloses such beads coated with a specific ligand and specifies that the remaining active sites on the beads must be blocked prior to the assay to prevent the reactant of interest or others from binding directly to the beads rather than to the ligand. This disclosure leads away from the present invention.
Despite the wide applications of this technology since its inception, there has not been the slightest suggestion that this same technology could be used advantageously to detect radiolabeled phosphate through hydrophobic interaction with a phosphomolybdate complex. Applicant""s use of the SPA concept in the detection of ATPase activity is founded on the observation that the hydrophobic phosphomolybdate complex binds to hydrophobic surfaces, particularly to the surface of polyvinyl toluene SPA beads, whereas the charged ATP molecule does not. Applicant has used that property to separate the orthophosphate from ATP or ADP and takes advantage of the scintillant-coated beads for measurement of radioactive orthophosphate. Applicant therefore provides a robust method for detecting and measuring orthophosphate. This assay is amenable to large scale and provides reproducible results for detection of Pi in the low nanomolar range. This method is also suitable for kinetic analysis not easily performed by prior art assays.
The present invention uses the principle that phosphomolybdate binds to hydrophobic surfaces to isolate the phosphomolybdate complex from other phosphate-containing molecules and further uses the SPA concept to bring a radiolabeled phosphomolybdate complex in close contact with a scintillant for measurement by scintillation counting.
In a first embodiment, the present invention provides a method for detecting and measuring radiolabeled orthophosphate (Pi) in an aqueous reaction mixture, comprising the steps of:
a. adding a solution of molybdate to said reaction mixture under acidic conditions to form a phosphomolybdate complex; and
b. contacting said phosphomolybdate complex with a scintillant hydrophobic surface;
whereby binding of phosphomolybdate to the surface provides enough proximity for the radiolabeled phosphate to induce measurable scintillation of the scintillant correlating the amount of orthophosphate.
In a second embodiment, the present invention consists of a method for determining ATPase activity, comprising the steps of:
a. mixing radiolabeled [xcex3-33P]ATP with an ATP hydrolyzing enzyme;
b. incubating reaction mixture a sufficient time to afford orthophosphate to be released from hydrolysis;
c. adding a solution of molybdate to said reaction mixture to form a phosphomolybdate complex;
d. contacting said phosphomolybdate complex with a scintillant hydrophobic surface; and
e. measuring scintillation of said scintillant as a means to calculate the amount of said orthophosphate.
Optionally, the method also comprises: step f) adding a solution of CsCl to said reaction mixture prior to counting. Further optionally, the method comprises step g) adding a solution of citric acid to said CsCl-containing mixture prior to counting.
In a third embodiment, the present invention consists of an assay for screening inhibitors of a phosphate-hydrolyzing enzyme activity comprising the steps of: carrying out steps a to e (and optionally steps f and g) according to the method above, in the presence and absence of a candidate inhibitor; and comparing the amounts of orthophosphate in each case to calculate the levels of inhibition.
Commercially available SPA beads are microscopic beads impregnated with scintillant, and are available with a variety of molecules attached to their surface (e.g. streptavidin, glutathione, protein A). Polyvinyl toluene (PVT) SPA beads have relatively hydrophobic surfaces, and are capable of selectively adsorbing phosphomolybdate from reaction mixtures in the presence of excess ATP. Although SPA beads are treated to make them less hydrophobic, the hydrophobic interaction may be enhanced by high concentrations of cesium chloride commonly used to float beads in SPA protocols. In an aqueous medium, weak xcex2-particle emitters such as [33P] need to be in close physical proximity to scintillant molecules to cause them to emit lightxe2x80x94otherwise their energy is dissipated and lost in the solvent. Thus, [33P]-labeled phosphate complexed with molybdate and bound to the bead surface causes activation of the scintillant, whereas [33P]-labeled ATP free in solution does not. The light emitted by a sample is measured by a xcex2 scintillation counter and is proportional to the amount of phosphate present. SPA beads are commercially available and are presently treated by the company with a polyhydroxy film to be less hydrophobic. It is however contemplated by the Applicant that non-treated SPA beads may be particularly suitable for this particular assay.
The method and assays of the present invention are useful not only for the HPV E1 helicase, but for any ATPase, NTPase, or any enzyme which generates orthophosphate as a product, especially if the substrate Km is in the nanomolar to low micromolar range.
Particularly, the assay of the present invention is useful for determining the ATPase activity of various ATP hydrolyzing enzymes where it is desirable to run the assay at nM to low xcexcM concentrations of substrate. Such enzymes include (without being restricted thereto): helicases (e.g. from other viruses such as HPV, HSV, CMV, HCV), other infectious agents (e.g. bacteria), or cellular helicases; other energy transducing ATPases (such as for example myosins, dyneins, kinesins), ion transport ATPases, or chaperoning; other nucleotide phosphate-hydrolyzing enzymes (e.g. G proteins); protein or small molecule phosphatases; or inorganic pyrophosphatases.
In a fourth embodiment, the present invention comprises a kit for measuring radiolabeled orthophosphate in an aqueous solution, said kit comprising:
a. a solution of molybdate; and
b. a scintillant hydrophobic surface;
wherein said molybdate solution is added to said aqueous solution to form a phosphomolybdate complex, said complex being captured by said hydrophobic surface to induce measurable scintillation thereto.
A number of documents are cited in the present application. The content of such citations is incorporated herein by reference.
Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of the preferred embodiments with reference to the accompanying drawings which is exemplary and should not be interpreted as limiting the scope of the invention.